Patch-Clamp Investigations and Compartmental Modeling of Rod Bipolar Axon Terminals in an In Vitro Thin-Slice Preparation of the Mammalian Retina

Abstract

Oltedal L, Mørkve SH, Veruki ML, Hartveit E. Patch-clamp investigations and compartmental modeling of rod bipolar axon terminals in an in vitro thin-slice preparation of the mammalian retina. J Neurophysiol 97: [1171][1172][1173][1174][1175][1176][1177][1178][1179][1180][1181][1182][1183][1184][1185][1186][1187] 2007. First published December 13, 2006; doi:10.1152/jn.01010.2006. To extend the usefulness of rod bipolar cells for studies of chemical synaptic transmission, we have performed electrophysiological recordings from rod bipolar axon terminals in an in vitro slice preparation of the rat retina. Whole cell recordings from axon terminals and cell bodies were used to investigate the passive membrane properties of rod bipolar cells and analyzed with a two-compartment equivalent electrical circuit model developed by Mennerick et al. For both terminal-and soma-end recordings, capacitive current decays were well fitted by biexponential functions. Computer simulations of simplified models of rod bipolar cells demonstrated that estimates of the capacitance of the axon terminal compartment can depend critically on the recording location, with terminal-end recordings giving the best estimates. Computer simulations and whole cell recordings demonstrated that terminal-end recordings can yield more accurate estimates of the peak amplitude and kinetic properties of postsynaptic currents generated at the axon terminals due to increased electrotonic filtering of these currents when recorded at the soma. Finally, we present whole cell and outside-out patch recordings from axon terminals with responses evoked by GABA and glycine, spontaneous inhibitory postsynaptic currents, voltage-gated Ca 2ϩ currents, and depolarization-evoked reciprocal synaptic responses, verifying that the recorded axon terminals are involved in normal pre-and postsynaptic relationships. These results demonstrate that axon terminals of rod bipolar cells are directly accessible to whole cell and outside-out patch recordings, extending the usefulness of this preparation for detailed studies of pre-and postsynaptic mechanisms of synaptic transmission in the CNS.

Calcium spike-mediated digital signaling increases glutamate output at the visual threshold of retinal bipolar cells

“…During somatic recordings, amplitude and rise time of fast (synaptic) currents, generated at the axon terminal of mammalian RBCs, are significantly reduced because of filtering by the axonal resistance and somatic C m (Oltedal et al 2007). This might explain why fast Ca 2ϩ spikes were not reported during decades of electrophysiological investigations of BCs in mammalian retinas.…”

“…Coincidentally, in vivo imaging of transgenic zebrafish retinas expressing Ca 2ϩ reporters described multiple types of Ca 2ϩ spike-producing BCs in addition to Mbs Dreosti et al 2011). Although light-triggered Ca 2ϩ spikes in Mbs are robust enough for electrophysiological detection via somatic recordings Protti et al 2000), we recorded light responses directly from Mb axon terminals to minimize filtering Oltedal et al 2007). By voltage clamping axotomized Mb terminals, free of space-clamp errors (Mennerick et al 1997;Oltedal et al 2007), at previously recorded light-evoked V m , we obtained precise and realistic measures of I Ca and glutamate release, reflected by C m increase, from Mb terminals in response to a natural stimulus: light.…”

“…Axotomized BC terminals offer a perfect presynaptic structure for voltage-clamped current and C m measurements with high temporal resolution (Mennerick et al 1997;Oltedal et al 2007;Palmer et al 2003). We used the light-evoked V m waveforms, previously recorded from the axon terminal of intact Mbs, as command potentials in voltage-clamp protocols to trigger I Ca and consecutive glutamate release in axotomized Mb terminals.…”

“…Furthermore, Mb axon terminals are large enough for direct measurement of exocytosis-associated increases in their membrane capacitance (C m ) with high fidelity (von Gersdorff and Matthews 1999). Although the complex morphology of intact BCs can be considered during C m measurements (Mennerick et al 1997;Oltedal et al 2007), the slowly decaying light-evoked synaptic currents (Saito et al 1979) might prevent a reliable measurement of C m changes (Gillis 2000) associated with light responses of Mbs. Therefore, first, light-evoked membrane potential (V m ) changes were recorded directly from the axon terminals of intact Mbs.…”

Regulation of ON bipolar cell activity

“…By comparison, the single photon response, measured in voltage clamp, has been estimated to be between 5 and 10 pA in mouse (Field and Rieke, 2002;Berntson et al, 2004b), similar to the estimate of 8 pA in dogfish, obtained 20 years earlier (Ashmore and Falk, 1982) using sharp electrode recording and noise analysis. For a rod bipolar cell with an input resistance of 2 GΩ (Zhou et al, 2006;Oltedal et al, 2007), the single photon response would produce a depolarization of approximately 10-20 mV in the rod bipolar cell. A response of this magnitude, rising from a baseline membrane potential of −55 mV is well matched to the voltage required for L-type Ca 2+ channel activation.…”